Acireductone dioxygenase (ARD) is found in the ubiquitous methionine salvage pathway (MSP) in animals, plants, and bacteria. Enzymatically it is implicated in the regulation of SAM and MTA; the latter is a result of polyamine synthesis and a regulator of cell growth in animals. The enzyme can bind to iron and nickel giving two distinct products. The nickel reaction (?off-pathway?) catalyzes the transformation of acireductone into 3- (methylthio)propionate, formate and carbon monoxide, a known anti-apoptotic signaling molecule. ARD is the only known example of a metalloenzyme whose function differs only by the identity of the metal ion. Recent mammalian studies have characterized mouse (MmARD) and human analogues (HsARD) of the enzyme. In mammalian systems it has been shown that ARD is capable of binding manganese and cobalt to perform ?off- pathway? type chemistry. Recent work with these analogues in in-vitro studies has shown that HsARD might play an intracellular regulatory role in brain tumors. Furthermore, it has been shown that the gene coding for HsARD, ADI1, is downregulated in human and rat prostate cancer cells as well as gastrocarcinoma and fibrosarcoma cells. These moonlighting functions, as well as the enzymatic roles of ARD beg the question about the connection between ARD in disease. To date there is still debate about the exact mechanism of the regiospecific substrate oxidation, and little is known about the role that metal identity plays in this reactivity. This project uses synthetic biomimetic modeling to contribute to answering these questions. The proposed work aims at greatly expanding the availability of models of ARD using nickel and mammalian relevant metals with two specific aims: 1. Expand the limited availability of biomimetic structural and functional models of Ni- ARD. These will be used to test the structural and mechanistic reasons for the observed ?off-pathway? oxidative regioselective reactivity in ARD. Mechanistic and kinetic studies will follow to provide valuable information to explain the mechanism of regioselective substrate activation in Ni-ARD. 2. Synthesize and begin to study the biomimetic reactivity of model complexes using metals relevant to mammalian systems (cobalt and manganese specifically). These new compounds will contribute to the understanding of the ?off-pathway? reactions and the role of metal identity in promoting disease in human cells.
Our proposed project is relevant to NIH?s mission because very recent cell and genetic studies have implicated acireductone dioxygenase (ARD) in cell proliferation, tumor regulation, and disease. In humans it is hypothesized that moonlighting, metal dependent, reactivity in non-native environments might be responsible for the observed oncogenic/disease connection. Our project aims to understand how this metal dependency might be implicated in promoting disease by using model compounds to guide our understanding of the mechanism ARD.